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Fluorescently-Labeled Glutathiones to Quantify Cysteine Reactivity
Patolia, Saloni H
Patolia, Saloni H
Abstract
The purpose of this study was to quantify cysteine reactivity with fluorescently-labeled glutathiones. Previous work on fluorescein-labeled glutathiones had established that they undergo S-glutathionylation with GAPDH and tubulin cysteines. In the current study, the proteins GAPDH and LDH were reacted with fluorescein-labeled and dansyl-labeled glutathiones under different reaction conditions identical to steric, salt, and pH environments in a cell. These reactions were monitored via UV/Vis spectroscopy to obtain quantitative data on reactivity of GAPDH and LDH cysteines. Reactions were determined qualitatively through nitrocellulose membrane dot blots and SDS-PAGE gels. Cysteine reactivity with fluorescently- labeled glutathiones was shown to be influenced by sterics, salt concentration, and pH. Reactivity was higher in conditions of low steric hindrance and in high salt concentration. Interestingly, reactivity was decreased at a pH higher than physiological pH. By establishing that the change in reactivity of protein cysteines in various cellular conditions can be investigated via reaction with fluorescent glutathiones and subsequent monitoring by UV/Vis spectroscopy, I have demonstrated that cysteine reactivity of the proteins GAPDH and LDH can be quantified by fluorescently-labeled glutathiones.
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2017-05-01
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Chemistry